The mitochondrial dysfunction observed in the pif1 knockout mouse, coupled with the enrichment of rare pif1 variants in the human mitochondrial disease population, suggest that PIF1 activity is essential for maintaining mitochondrial respiratory function and the prevention of disease. Our objective is to identify the crucial mitochondrial PIF1 functions that normally prevent juvenile complex I deficiency and age-related formation of mitochondrial DNA (mtDNA) deletions to expand our understanding of the role of PIF1 in human health. PIF1 is a G-quadruplex (GQ) helicase that localizes to the nucleus and mitochondria. G-quadruplexes are guanine-rich secondary structures that form in nucleic acids, and in the nucleus cause DNA instability and interfere with translation. We recently detected GQ in the mitochondria of cultured cells by immunocytochemistry, raising the exciting possibility that these structures could play a role in the development of mitochondrial dysfunction. Supporting our idea that GQ play a role in human mitochondrial disease, we recently showed that the location of human mtDNA deletion breakpoints associates significantly with G-quadruplexes. We hypothesize that the loss of the GQ resolution in mitochondrial nucleic acids contributes to the deficits in mitochondrial respiration and the mtDNA instability seen in the pif1 null mouse. We will test this hypothesis using complementation assays in pif1 yeast, cultured pif1 null mouse cells, and KO skeletal muscle. We propose to target well defined DNA- and RNA-directed GQ and non-GQ helicases to pif1-deficient mitochondria to identify the activities required for complex I activity and mtDNA stability. Strong candidates will then be tested in mouse pif1 KO skeletal muscle in vivo using adeno-associated virus (AAV) delivery. We will also identify any defective steps in mitochondrial protein production by examining pif1 null and helicase-complemented cells for complex I transcript abundance, translation, and respiratory complex assembly. The relationship between GQ abundance and mtDNA deletion load in WT, null, and helicase-complimented muscle will be determined by coupling immunodetection of GQ with next generation sequencing. This strategy will also identify associations between GQ position and deletion breakpoint location in pif1 null and rescued samples. These mechanistic studies will support our pursuit of PIF1 as a human disease gene in patients with documented mtDNA deletions or complex I deficiency. Importantly, we have identified rare PIF1 homozygous and compound heterozygous mutations in human patients with mitochondrial disease, which highlights PIF1 as a potential human disease gene. The balance between mitochondrial GQ structure formation and PIF1 activity offers novel yet highly promising insight into human disease, and the experiments proposed here have the potential to open a new field of research in mitochondrial medicine.